Quantitative assessment of human serum high-abundance protein depletion.
The aim of this study is to quantify the effectivity of the depletion of human high-abundance serum and plasma proteins for improved protein identification and disease marker candidate discovery and to assess the risk of concomitant removal of relevant marker proteins. 2-DE and bottom-up shotgun MS combining 2-D capillary chromatography with MS/MS were applied in parallel for the analysis of fractions resulting from the depletion procedure. For many proteins the factors of enrichment by the depletion were obvious allowing their enhanced detection and identification upon high-abundance protein depletion. Nano-liquid chromatography linked MS allowed the efficient identification of several low-abundant proteins that were not identified on the 2-DE gels. Resolving the fractions that were eluted from the matrix upon depletion indicated unspecific binding of disease relevant proteins in plasma samples from acute myocardial infarction patients. The unspecific binding to the depletion matrix of inflammatory markers spiked into the serum was found to depend on the type of capturing agent used. Polyclonal avian antibodies (IgY) displayed the least unspecific binding due to the high immunogenicity of mammalian proteins in avian hosts.
Stempfer
Rene
R
Molecular Diagnostics, Life Sciences, Austrian Research Centers GmbH - ARC, Seibersdorf, Austria.
Kubicek
Markus
M
Lang
Irene M
IM
Christa
Noehammer
N
Gerner
Christopher
C
D001798:Blood Proteins / Q000032:analysis*; D002853:Chromatography, Liquid; D016207:Cytokines / Q000097:blood; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008297:Male; D011485:Protein Binding; D020543:Proteome / Q000032:analysis*; D011994:Recombinant Proteins / Q000097:blood; D044967:Serum; D053719:Tandem Mass Spectrometry
D001798:Blood Proteins; D016207:Cytokines; D020543:Proteome; D011994:Recombinant Proteins